EP3047275B1 - Compositions et procédés permettant de renforcer la migration de cellules souches mésenchymateuses en direction de tumeurs - Google Patents

Compositions et procédés permettant de renforcer la migration de cellules souches mésenchymateuses en direction de tumeurs Download PDF

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EP3047275B1
EP3047275B1 EP14842369.2A EP14842369A EP3047275B1 EP 3047275 B1 EP3047275 B1 EP 3047275B1 EP 14842369 A EP14842369 A EP 14842369A EP 3047275 B1 EP3047275 B1 EP 3047275B1
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mscs
cells
tumor
msc
amf
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EP3047275A4 (fr
EP3047275A1 (fr
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Guillermo Daniel MAZZOLINI
Mariana Gabriela GARCIA
Juan BAYO
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Asociacion Civil De Estudios Superiores
Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Inis Biotech LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • MSCs are most often derived from bone marrow (BM), but can also be isolated from adipose tissue (AT) or from umbilical cord; from the latter case, MSC are isolated from the Wharton's jelly (WJ-MSCs), perivascular areas (mesenchymal cells harvested from umbilical cord perivascular tissue) or umbilical cord blood (CB-MSCs) ( Bernardo, M. E., F. Locatelli, et al. (2009) Ann N Y Acad Sci 1176: 101-117 ).
  • WJ-MSCs Wharton's jelly
  • perivascular areas mesenchymal cells harvested from umbilical cord perivascular tissue
  • CB-MSCs umbilical cord blood
  • MSCs show tropism for inflamed, injured or tumorigenic sites and their ability to be cultured and expanded in vitro, their self-renewal properties and low immunogenicity make these cells useful for cell therapy ( Prockop, D. J. and J. Y. Oh (2012) J Cell Biochem 113(5): 1460-1469 ).
  • HCC hepatocellular carcinoma
  • Autocrine motility factor is a 55-kDa cytokine (SEQ ID NO:1) secreted by tumors that regulates cell motility ( Liotta, L. A., R. Mandler, et al. (1986) Proc Natl Acad Sci U S A 83(10): 3302-3306 ).
  • AMF was isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells (Liotta, L. A., R. Mandler, et al. (1986)).
  • AMF exhibits sequence identity with glucose-6-phosphate isomerase (GPI) (alternatively known as phosphoglucose isomerase or phosphohexose isomerase (PHI)), a glycolytic enzyme involved in carbohydrate metabolism ( Watanabe, H., K. Takehana, et al. (1996) Cancer Res 56(13): 2960-2963 ).
  • GPI glucose-6-phosphate isomerase
  • PHI phosphohexose isomerase
  • the stimulation of cell motility is induced by the binding to the autocrine motility factor receptor (AMFR), a 78-kDa seven transmembrane glycoprotein with leucine zipper and RING-H2 motifs ( Shimizu, K., M. Tani, et al. (1999) FEBS Lett 456(2): 295-300 ).
  • AMFR is stably localized in caveolae
  • caveolin-1 Cav-1
  • Cav-1 has the ability to regulate the endocytic pathway through the stabilization of caveolae expression
  • AMF is secreted by different tumors such as lung ( Dobashi, Y., H. Watanabe, et al. (2006) J Pathol 210(4): 431-440 ), gastrointestinal, kidney and mammary ( Baumann, M., A. Kappl, et al. (1990) Cancer Invest 8(3-4): 351-356 ) as well as by hepatocellular carcinomas ( Torimura, T., T. Ueno, et al. (2001) Hepatology 34(1): 62-71 ). Migration of hepatocellular carcinoma cells upon AMF stimulation has been associated to upregulation of metalloproteinase 3 (MMP3) ( Yu, F. L., M. H. Liao, et al.
  • MMP3 metalloproteinase 3
  • Hepatocellular carcinoma is the sixth most common cancer worldwide and the third cause of cancer-related death ( Ferenci, P., M. Fried, et al. (2010) J Gastrointestin Liver Dis 19(3): 311-317 ). Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis. Curative therapies such as resection or liver transplantation have been demonstrated to improve patient survival ( de Lope, C. R., S. Tremosini, et al. (2012) J Hepatol 56 Suppl 1: S75-87 ); however, these strategies can only be applied to a minority of patients. Therefore, there is an urgent therapeutic need for patients with HCC.
  • hepatitis B or C hepatitis B or C
  • Curative therapies such as resection or liver transplantation have been demonstrated to improve patient survival ( de Lope, C. R., S. Tremosini, et al. (2012) J Hepatol 56 Suppl 1: S75-87
  • the present invention provides a method for (1) increasing migration of a mesenchymal stromal cell (MSC) to a tumor or a tumor cell or (2) for increasing adhesion of an MSC to an endothelial cell, wherein the method comprises stimulating the MSC with a recombinant autocrine motility factor (rAMF) in vitro, wherein the MSC comprises a therapeutic agent.
  • MSC mesenchymal stromal cell
  • rAMF autocrine motility factor
  • isolated in regard to cells, refers to a cell that is removed from its natural environment (such as in a solid tumor) and that is isolated or separated, and is at least about 30% free, about 50% free, about 75% free, about 90% free, about 95% free, or 100% free, from other cells with which it is naturally present, but which lack the marker based on which the cells were isolated.
  • heterologous refers to, e.g., a gene, polypeptide or cell that is not in its natural environment; thus, it is non-naturally-occurring.
  • a heterologous gene or polypeptide includes a gene or polypeptide from one species introduced into another species.
  • a heterologous gene or polypeptide also includes a gene or polypeptide native to an organism that has been altered in some way ( e.g ., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.).
  • a heterologous cell includes a cell native to an organism that has been altered in some way (e.g ., genetically modified to include a recombinant gene, protein, or virus).
  • Tumor and neoplasm refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
  • cancer refers to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include liver cancer (e.g ., hepatocellular carcinoma (HCC)), colon cancer, colorectal cancer (e.g ., colorectal carcinoma), gastrointestinal cancer, pancreatic cancer, lung cancer, breast cancer, and kidney cancer.
  • HCC hepatocellular carcinoma
  • colorectal cancer e.g ., colorectal carcinoma
  • Metalastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
  • the term “subject” refers to any animal (e.g ., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • the term "subject suspected of having cancer” refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass) or is being screened for a cancer ( e.g ., during a routine physical).
  • a subject suspected of having cancer can also have one or more risk factors.
  • a subject suspected of having cancer has generally not been tested for cancer.
  • a "subject suspected of having cancer” encompasses an individual who has received an initial diagnosis but for whom the stage of cancer is not known. The term further includes people who once had cancer ( e.g., an individual in remission).
  • the term "subject at risk for cancer” refers to a subject with one or more risk factors for developing a specific cancer.
  • Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, previous incidents of cancer, pre-existing non-cancer diseases, and lifestyle.
  • the term "subject diagnosed with a cancer” refers to a subject who has been tested and found to have cancerous cells.
  • the cancer can be diagnosed using any suitable method, including but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present disclosure.
  • the term "characterizing cancer in a subject” refers to the identification of one or more properties of a cancer sample in a subject, including but not limited to, the presence of benign, pre-cancerous or cancerous tissue, the stage of the cancer, and the subject's prognosis. Cancers can be characterized by the identification of the expression of one or more cancer marker genes, including but not limited to, any cancer markers disclosed herein.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (e.g., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (e.g., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
  • Molecules e.g., transcription factors
  • activators e.g., transcription factors
  • high levels increased levels
  • high expression increased expression
  • elevated levels increased levels
  • up-regulated expression in regard to gene expression are used herein interchangeably to refer to expression of a gene in a cell or population of cells at levels higher than the expression of that gene in a second cell or population of cells.
  • “high levels”, “increased levels”, “high expression”, “increased expression”, “elevated levels” or “up-regulated expression” can be determined by detecting the amount of a polynucleotide (mRNA, cDNA, etc.) in tumor cells, for example, by quantitative RT-PCR or microarray analysis; or by detecting the amount of a protein in tumor cells, for example, by ELISA, Western blot, quantitative immunofluorescence.
  • low levels As used herein, the terms “low levels”, “decreased levels”, “low expression”, “reduced expression” or “decreased expression” in regards to gene expression are used herein interchangeably to refer to expression of a gene in a cell or population of cells, at levels less than the expression of that gene in a second cell or population of cells.
  • Low levels of gene expression can be determined by detecting decreased to nearly undetectable amounts of a polynucleotide (mRNA, cDNA, etc.) in tumor cells, for example, by quantitative RT-PCR or microarray analysis.
  • “low levels” of gene expression can be determined by detecting decreased to nearly undetectable amounts of a protein in tumor cells, for example, ELISA, Western blot, or quantitative immunofluorescence.
  • detectable levels or “loss of expression” in regards to gene expression as used herein refers to expression of a gene in a cell or population of cells, at levels that cannot be distinguished from background using conventional techniques such that no expression is identified.
  • "Undetectable levels” of gene expression can be determined by the inability to detect levels of a polynucleotide (mRNA, cDNA, etc.) in tumor cells above background by, for example, quantitative RT-PCR or microarray analysis.
  • “undetectable levels” of gene expression can be determined by the inability to detect levels of a protein in tumor cells above background by, for example, ELISA, Western blot, or immunofluorescence.
  • nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA.
  • the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyla
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • a cDNA form of a gene is "intron-free" and non-naturally-occurring.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (e.g., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
  • Molecules e.g., transcription factors
  • activators e.g., transcription factors
  • isolated polypeptide refers to a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g ., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which can be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
  • heterologous polypeptide refers to a polypeptide that is not in its natural environment; thus, it is non-naturally-occurring.
  • a heterologous polypeptide includes a polypeptide from one species introduced into another species.
  • a heterologous polypeptide also includes a polypeptide native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-polypeptide, etc.).
  • Heterologous polypeptide are distinguished from endogenous polypeptide in that the heterologous polypeptide sequences are typically encoded by cDNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g ., genes expressed in loci where the gene is not normally expressed).
  • a mutation can be made by any technique for mutagenesis known in the art, including but not limited to, in vitro site-directed mutagenesis ( Hutchinson et al., J. Biol. Chem. 253:6551 (1978 ); Zoller et al., DNA 3:479 (1984 ); Oliphant et al., Gene 44:177 (1986 ); Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986 )), use of TAB® linkers (Pharmacia), restriction endonuclease digestion/fragment deletion and substitution, PCR-mediated/oligonucleotide-directed mutagenesis, and the like.
  • in vitro site-directed mutagenesis Hutchinson et al., J. Biol. Chem. 253:6551 (1978 ); Zoller et al., DNA 3:479 (1984 ); Oliphant et al., Gene 44:177 (1986
  • PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70 ).
  • variants can include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin.
  • the techniques for obtaining non-naturally-occurring variants including genetic (suppressions, deletions, mutations, etc. ) , chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • sequence analysis software refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences.
  • Sequence analysis software can be commercially available or independently developed. Typical sequence analysis software includes, but is not limited to, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX ( Altschul et al., J. Mol. Biol. 215:403 (1990 )), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA).
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • In vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g ., an animal or a cell) that can be, but are not limited to, processes or reaction that occur within a natural environment.
  • ex vivo refers to "outside” the body.
  • in vitro can be used interchangeably herein.
  • MSC Mesenchymal stromal cell
  • MSCs Mesenchymal stromal cells
  • fibroblasts also referred to as fibroblastic colony forming units or mesenchymal stem cells
  • MSCs Mesenchymal stromal cells
  • MSCs are most often derived from bone marrow (BM), but can also be isolated from adipose tissue (AT) or from umbilical cord (youngest, most primitive MSCs); from the latter case, MSCs are isolated from the Wharton's jelly (WJ-MSCs), perivascular areas (Mesenchymal cells harvested from umbilical cord perivascular tissue) or umbilical cord blood (CB-MSCs) ( Bernardo, M. E., F. Locatelli, et al. (2009) Ann N Y Acad Sci 1176: 101-117 ). Other rich sources for MSCs are the developing tooth bud of the mandibular third molar and amniotic fluid.
  • WJ-MSCs Wharton's jelly
  • CB-MSCs umbilical cord blood
  • MSCs can be successfully isolated from human peripheral blood ( Chong PP et al. (2012) J Orthop Res. 30(4):634-42 ).
  • the MSCs can be isolated from the whole umbilical cord and in this case can be referred to as "mesenchymal cells derived from umbilical cord.” It is noted that as the umbilical cord has different structures, and the isolation of MSCs can also be made or harvested from only a "region" or "structure” such as the perivascular tissue, the Wharton's jelly, or the umbilical cord blood.
  • MCSs have been observed to have anti-inflammatory effects.
  • the disease models in which MSCs have produced beneficial effects include diabetes, stroke, spinal cord injury, Parkinsonism, Alzheimer's disease, liver disease, kidney disease, and some cancers. See Prockop, D. J. and J. Y. Oh (2012) J Cell Biochem 113(5): 1460-1469 .
  • MSCs have also been shown to contribute to cancer progression, e.g., hematological malignancies ( Torsvik A. and Bjerkvig R. (2013) Cancer Treat Rev. 39(2)180-8 ).
  • MSCs have the ability to migrate and engraft tumors and it is thought that factors produced by tumor cells and their microenvironments are responsible.
  • MSC motility in vitro has been induced after stimulation with different cytokines ( Ries, Egea et al. (2007). Blood 109(9): 4055-4063 ), growth factors ( Ponte, Marais et al. (2007) Stem Cells 25(7): 1737-1745 ), or chemokines such as CXCL7 ( Kalwitz, Endres et al. (2009) Int J Biochem Cell Biol 41(3): 649-658 ) or SDF-1 ( Gao, Priebe et al. (2009) Stem Cells 27(4): 857-865 ).
  • MSCs show tropism for inflamed, injured or tumorigenic sites and their ability to be cultured and expanded in vitro, their self-renewal properties and low immunogenicity make these cells useful for cell therapy ( Prockop, D. J. and J. Y. Oh (2012) J Cell Biochem 113(5): 1460-1469 ). Although there are some promising results with MSCs genetically modified as a therapeutic option for HCC ( Gao, Yao et al. (2010) Oncogene 29(19): 2784-2794 ; Niess, Bao et al.
  • MSCs e.g., MSCs genetically modified to express an anti-tumor gene
  • rAMF rAMF
  • a method for the genetic modification of MSCs is by chemical (e.g . Lipofectamine) or physical (e.g. electroporation) transfection or viral vectors. Afterwards stably transfected cells can be selected, where the transgene cassette has integrated by chance into the MSC genome.
  • the genetic modification of MSCs is by using non-viral vector systems derived from transposons. After flanking of an expression cassette with terminal inverted repeats, a construct can be transferred into MSC via transfection. If a transposase is expressed in trans during the transfection, the expression cassette will be stably integrated into the genome of the MSC.
  • a genetically modified MSC can be prepared by transduction of native MSCs with pseudotyped virions, expressing foreign glycoproteins on their surface, which alter the tropism and often the titer of the virion.
  • a genetically modified MSC can be engineered to express an oncolytic virus expressing anti-tumor genes.
  • Autocrine motility factor is a 55-kDa cytokine secreted by tumors that regulates cell motility ( Liotta, L. A., R. Mandler, et al. (1986) Proc Natl Acad Sci U S A 83(10): 3302-3306 ).
  • AMF exhibits sequence identity with glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in carbohydrate metabolism ( Watanabe, H., K. Takehana, et al. (1996) Cancer Res 56(13): 2960-2963 ).
  • GPI glucose-6-phosphate isomerase
  • AMFR autocrine motility factor receptor
  • Cav-1 caveolin-1
  • AMF-induced motility is mediated by upregulation of MMP2 and MMP3 ( Torimura, Ueno et al. (2001) Hepatology 34(1): 62-71 ; Yu, Liao et al. (2004) Biochem Biophys Res Commun 314(1): 76-82 ).
  • AMF was shown to increase the expression of mRNA MMP3 in MSCs. It was previously reported that MSCs exposed to CM derived from HCC cell lines increased their MMP2 activity ( Garcia, Bayo et al. (2011) Mol Pharm 8(5): 1538-1548 ).
  • AMF is produced by several tumors, such as lung ( Dobashi, Watanabe et al. (2006) J Pathol 210(4):431-440 ), gastrointestinal, kidney and breast ( Baumann, Kappl et al. (1990) Cancer Invest 8(3-4):351-356 as well as hepatocellular carcinomas (HCC) ( Ogata, Torimura et al. (1999) Hum Pathol 30(4): 443-450 ). It is also reported herein that AMF is secreted in the CM from HCC s.c tumors.
  • AMF is not considered a typical chemotactic factor such as VEGF, PDGF, TGF- ⁇ , MCP-1, IL-8, TNF- ⁇ , IL-1 ⁇ , IL-6, SDF-1, and HGF. Instead, intracellular AMF has been shown to be involved in glucose metabolism in all types of cells and some reports have described the extracellular form of AMF as inducing tumor migration and endothelial cell migration related to angiogenesis.
  • AMF-induced migration has been described in tumor cells and its role in metastasis.
  • exogenous AMF stimulated migration of human cancer melanoma, fibrosarcoma and HCC cells as well as human umbilical vein endothelial cells (HUVECs)
  • UAVECs human umbilical vein endothelial cells
  • rAMF treatment does not induce malignant transformation in MSCs or promote increased tumor development or metastasis.
  • AMF-induced migration is mediated by its interaction with AMF receptor (AMFR) on cell surface ( Silletti, Watanabe et al. (1991) Cancer Res 51(13): 3507-3511 ).
  • AMFR AMF receptor
  • rAMF treatment of MSCs induced AMFR and caveolin-1 and -2 expressions, supporting their role in the maintenance of the receptor on the cell surface.
  • AMF enhances integrin ⁇ 1 activity leading to activation of mitogen activated protein kinase (MAPK) and Rho pathways ( Torimura, Ueno et al. (2001) Hepatology 34(1): 62-71 ).
  • MME mitogen activated protein kinase
  • Rho pathways Torimura, Ueno et al. (2001) Hepatology 34(1): 62-71 .
  • Small GTPase is largely involved in motility and cell adhesion due to its role in cytoskeleton organization.
  • GTPase activity is regulated by GTPase-activating proteins (GAPs) and GDP dissociation inhibitors (GDIs).
  • Rho GDP dissociation inhibitor (GDI) ⁇ (GDI2) is diminished in cells with higher motility indicating its role as suppressor of migration.
  • GDI2 Rho GDP dissociation inhibitor
  • rAMF treatment decreased mRNA of GDI-2, supporting its role as inhibitor of migration.
  • rAMF is a chemoattractant factor for MSCs, e.g., MSCs comprising a therapeutic agent.
  • the AMF can be naturally produced or recombinant.
  • the AMF is human AMF.
  • the AMF comprises the polypeptide sequence of SEQ ID NO:1 or a functional fragment thereof.
  • the AMF comprises a polypeptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an AMF amino acid sequence that is naturally produced in an animal ( e.g ., SEQ ID NO:1).
  • the AMF comprises or consists of a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:1 or a functional fragment thereof.
  • Certain embodiments are directed to a composition
  • a composition comprising a mesenchymal stromal cell (MSC) stimulated with a recombinant autocrine motility factor (rAMF), wherein the MSC comprises a therapeutic agent and wherein the MSC of the composition has (1) increased migration to a tumor or a tumor cell after rAMF stimulation and/or (2) increased adhesion to an endothelial cell, e.g., a vascular endothelial cell, after rAMF stimulation.
  • MSC mesenchymal stromal cell
  • rAMF recombinant autocrine motility factor
  • Certain embodiments are directed to a composition
  • a composition comprising a mesenchymal stromal cell (MSC) stimulated with a recombinant autocrine motility factor (rAMF), wherein the MSC comprises a therapeutic agent and wherein the MSC of the composition has increased migration to a tumor or a tumor-derived cell.
  • the increased migration is relative to or compared to MSC without rAMF stimulation.
  • Certain embodiments are directed to a composition
  • a composition comprising a mesenchymal stromal cell (MSC) stimulated with a recombinant autocrine motility factor (AMF), wherein the MSC comprises a therapeutic agent and wherein the MSC of the composition has increased adhesion to an endothelial cell, e.g., a vascular endothelial cell.
  • the increased adhesion is relative to or compared to MSC without rAMF stimulation.
  • the increased migration and/or adhesion is about 1.5-fold, about 2-fold, about 2.5-fold, or about 3-fold greater than migration and/or adhesion of the MSC without rAMF stimulation. In some embodiments, the increase in migration and/or adhesion is at least about 20-60%, 30-60%, 40-60%, 30-50%, or 20-40% greater than migration and/or adhesion of the MSC without rAMF stimulation.
  • the MSC of the composition is selected from the group consisting of bone marrow MSC, adipose tissue MSC, umbilical cord MSC, and any combination thereof.
  • the tumor is a solid tumor or cancer.
  • the tumor is a liver cancer, a colon cancer, a pancreatic cancer, a lung cancer, a gastrointestinal cancer, a kidney cancer, a breast cancer, or a combination thereof.
  • the tumor is a carcinoma, e.g ., hepatocellular carcinoma (HCC) or colorectal carcinoma.
  • the tumor or the tumor cell expresses endogenous AMF.
  • the therapeutic agent is a recombinant anti-tumor gene (e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), a suicide gene (e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha), or any combination thereof), a cytotoxic drug, an antibody, or an oncolytic virus.
  • an interferon e.g., interferon a, interferon ⁇
  • an interleukin interleukin 1, interleukin 12
  • chemokine e.g., CX3CL1
  • suicide gene e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha
  • the therapeutic agent is an oncolytic virus (OV), i.e., a virus that preferentially infects and kills cancer cells (e.g ., adenovirus (e.g., H101), Reovirus, measles, herpes simplex (e.g., HSV1716), Newcastle disease virus and vaccinia).
  • OV oncolytic virus
  • the oncolytic virus is engineered to expresses a recombinant anti-tumor gene.
  • the recombinant oncolytic virus is an oncolytic adenovirus.
  • the therapeutic agent is an oncolytic virus (see, e.g., Dwyer RM et al. (2010) Stem Cell Res Ther. 1(3):25 ), e.g., onyx-015 (see, e.g., Khuri FR et al. (2000) Nat Med. 6(8):879-85 ); Ad-F512(H-N)5/3 (see, e.g., Viale D.L., et al. (2013) J Invest Dermatol doi: 10.1038/jid.2013.191 (e-publication ahead of print)).
  • oncolytic virus see, e.g., Dwyer RM et al. (2010) Stem Cell Res Ther. 1(3):25 ), e.g., onyx-015 (see, e.g., Khuri FR et al. (2000) Nat Med. 6(8):879-85 ); Ad-F512(H-N)5/3 (see, e.g., Viale D
  • the oncolytic virus can be used as a vector for delivery of anti-tumor genes, e.g., an interferon (e.g ., interferon a, interferon ⁇ ), an interleukin (e.g., interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), or a suicide gene (e.g., encoding enzymes that can metabolize a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill neighboring cells).
  • the MSC comprises a recombinant AMF receptor, CXCR1, CXCR2, or MCP-1.
  • Certain instances are directed to a method for increasing migration or anchorage of a mesenchymal stromal cell (MSC) to a tumor comprising (a) stimulating the MSC with a recombinant autocrine motility factor (rAMF), and (b) administering the stimulated MSC of (a) to the tumor, wherein the MSC comprises a therapeutic agent.
  • MSC mesenchymal stromal cell
  • rAMF recombinant autocrine motility factor
  • the methods of the application include increasing migration or anchorage of MSCs to a tumor, e.g., a solid tumor or cancer.
  • the tumor is selected from the group consisting of a liver cancer, a colon cancer, a pancreatic cancer, a lung cancer, a gastrointestinal cancer, a kidney cancer, a breast cancer, and any combination thereof.
  • the tumor is a carcinoma, e.g ., hepatocellular carcinoma (HCC) or colorectal carcinoma.
  • the tumor or the tumor cell expresses endogenous AMF.
  • the methods of the application include increasing migration or anchorage of MSCs to a tumor wherein the MSCs comprise a therapeutic agent, e.g., a recombinant anti-tumor gene (e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), a suicide gene (e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha), or any combination thereof), a cytotoxic drug, an antibody, or an oncolytic virus.
  • a therapeutic agent e.g., a recombinant anti-tumor gene (e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (interleukin 1, interleukin 12), a chemokine (e.g.
  • the methods of the application include increasing migration or anchorage of MSCs to a tumor wherein the MSCs comprise a therapeutic agent, e.g., an oncolytic virus (OV), i.e., a virus that preferentially infects and kills cancer cells (e.g ., adenovirus (e.g., H101), Reovirus, measles, herpes simplex (e.g., HSV1716), Newcastle disease virus and vaccinia).
  • OV oncolytic virus
  • the oncolytic virus is engineered to expresses a recombinant anti-tumor gene.
  • the recombinant oncolytic virus is an oncolytic adenovirus, e.g., Ad-F512(H-N)5/3 ( see, e.g., Lopez, et al. (2012) Mol Ther. 20(12):2222-33 ).
  • the oncolytic virus can be used as a vector for delivery of anti-tumor genes, e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (e.g ., interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), or a suicide gene (e.g., encoding enzymes that can metabolize a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill neighboring cells).
  • the MSC comprises a recombinant AMF receptor, CXCR1, CXCR2, or MCP-1.
  • Certain aspects of the application are related to cell therapies using cells genetically engineered to express a heterologous gene, e.g., an anti-cancer gene. Some embodiments are directed to the composition of the application for use in a method for treating a subject with a tumor comprising administering to the subject the composition of the application.
  • a heterologous gene e.g., an anti-cancer gene.
  • Some embodiments comprise (a) stimulating a mesenchymal stromal cell (MSC) comprising a therapeutic agent with a recombinant autocrine motility factor (rAMF), and (b) administering the stimulated MSC of (a) to the subject.
  • MSC mesenchymal stromal cell
  • rAMF recombinant autocrine motility factor
  • Stimulating a MSC with a recombinant protein of the application, e.g ., rAMF is accomplished by pretreatment of the MSC prior to administration to a subject.
  • Methods for pretreating the MSC include, e.g ., culturing MSCs with the recombinant protein, e.g ., rAMF, for about 1-48 hours, about 6-48 hours, about 6-36 hours, about 6-24 hours, about 12-24 hours, about 12-36 hours, about 12-48 hours, about 18-48 hours, about 18-36 hours, or about 18-24 hours prior to administration of the stimulated MSC.
  • the subject's tumor is a solid tumor or cancer.
  • the tumor is selected from the group consisting of a liver cancer, a colon cancer, a pancreatic cancer, a lung cancer, a gastrointestinal cancer, a kidney cancer, a breast cancer, and any combination thereof.
  • the tumor is a carcinoma, e.g ., hepatocellular carcinoma (HCC) or colorectal carcinoma.
  • the tumor expresses endogenous AMF.
  • the tumor is metastatic and/or vascularized.
  • the MSCs comprise a therapeutic agent, e.g., a recombinant anti-tumor gene (e.g., an interferon (e.g ., interferon a, interferon ⁇ ), an interleukin (interleukin 1, interleukin 12), a chemokine (e.g ., CX3CL1), a suicide gene (e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha), or any combination thereof), a cytotoxic drug, an antibody, or an oncolytic virus.
  • a recombinant anti-tumor gene e.g., an interferon (e.g ., interferon a, interferon ⁇ ), an interleukin (interleukin 1, interleukin 12), a chemokine (e.g ., CX3CL1), a suicide gene (e.g., thymidine kinase
  • the therapeutic agent is an oncolytic virus, i.e., a virus that preferentially infects and kills cancer cells (e.g ., adenovirus (e.g., H101), Reovirus, measles, herpes simplex ( e.g., HSV1716), Newcastle disease virus and vaccinia).
  • adenovirus e.g., H101
  • Reovirus e.g., H101
  • measles e.g., HSV1716
  • Newcastle disease virus and vaccinia e.g., Nakashima et al. (2010) Cytokine Growth Factor Rev. 21(2-3):119-26 .
  • the oncolytic virus is engineered to expresses a recombinant anti-tumor gene.
  • the recombinant oncolytic virus is an oncolytic adenovirus, e.g., Ad-F512(H-N)5/3 ( see, e.g., Lopez, et al. (2012) Mol Ther. 20(12):2222-33 ).
  • Ad-F512(H-N)5/3 see, e.g., Lopez, et al. (2012) Mol Ther. 20(12):2222-33 ).
  • the oncolytic virus can be used as a vector for delivery of anti-tumor genes, e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (e.g., interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), or a suicide gene (e.g., encoding enzymes that can metabolize a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill neighboring cells).
  • an interferon e.g., interferon a, interferon ⁇
  • an interleukin e.g., interleukin 1, interleukin 12
  • chemokine e.g., CX3CL1
  • suicide gene e.g., encoding enzymes that can metabolize a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill
  • the suicide gene encodes Herpes simplex viral thymidine kinase
  • the subject ideally is treated with ganciclovir in a manner permitting the Herpes simplex viral thymidine kinase to render the ganciclovir cytotoxic.
  • Another possibility is the use of cytosine deaminase as a cytotoxic protein, which converts 5-fluorocytosine to the toxic compound 5-fluorouracil.
  • the method for treating a subject with a tumor comprises introducing into the subject's bloodstream a therapeutically effective amount of a rAMF stimulated MSC or composition of the application.
  • the administration to the subject is systemic (e.g., parenteral) or local, e.g., to an intra-hepatic artery.
  • the therapeutically effective number of MSCs includes, without limitation, the following amounts and ranges of amounts: (i) from about 1x10 5 to about 1x10 9 cells/kg body weight; (ii) from about 1x10 6 to about 1x10 8 cells/kg body weight; (iii) from about 5x10 6 to about 2x10 7 cells/kg body weight; (iv) from about 5x10 6 to about 1x10 7 cells/kg body weight; (v) from about 1x10 7 to about 2x10 7 cells/kg body weight; (vi) from about 7x10 6 to about 9x10 6 cells/kg body weight; (vii) about 1x10 5 cells/kg body weight; (viii) about 1x10 6 cells/kg body weight; (ix) about 5x10 6 cells/kg body weight; (x) about 1x10 7 cells/kg body weight; (xi) about 6x10 6 cells/kg body weight; (xii) about 7x10 6 cells/kg body weight; (xiii) about 8x
  • Human HCC cell line HuH7 were kindly provided by Prof. Jesus Prieto (CIMA, University of Navarra, Pamplona, Spain).
  • LX-2 cell line human HSCs generated by spontaneous immortalization in low serum conditions
  • HMEC-1 Human microvascular endothelial cells (HMEC-1) were provided by CDC (Centers for Disease Control, Atlanta, GA, USA).
  • Cell lines were cultured in complete DMEM (2 ⁇ mol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% heat-inactivated fetal bovine serum (FBS)).
  • FBS heat-inactivated fetal bovine serum
  • HCC cells Primary culture of HCC cells (HC-PT-5) was previously generated in our laboratory and cultured the eight passage in 70% DMEM/ 30% F12 (Invitrogen/Life Technologies) culture medium supplemented with 2 ⁇ mol/L glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin and 10 % FBS.
  • MSCs were characterized according to the guidelines from International society for cellular therapy (ISCT).
  • TCM tumor conditioned medium
  • HuH7 or HC-PT-5 subcutaneous tumors s.c.
  • s.c. subcutaneous tumors
  • BM-MSCs or AMF stimulated BM-MSC were lysed with 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.4, 0.1% SDS, 1.0% Nonidet P-40, 0.5% Na-deoxycholate, 0.2 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail. Lysates were centrifuged at 12,000 g for 20 min and the supernatants were used as total cell lysates. CCM and TCM were concentrated 100-fold using Vivaspin 6 centrifugal concentrator (Sartorius-Stedim Biotech). The protein concentration was determined by Bradford protein assay (Bio-Rad).
  • Protein was separated by SDS-PAGE and transferred onto nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). Blots were blocked and incubated with anti-AMF (1:700) polyclonal antibody (sc-33777, Santa Cruz Biotechnology), anti-AMFR (1:1000) polyclonal antibody (AP2162a, ABGENT), anti-JNK (1:1000) polyclonal antibody (9252, Cell Signaling), anti-phospho-JNK (1:1000) polyclonal antibody (9251, Cell Signaling), anti-c-Fos (1:1000) monoclonal antibody (2250, Cell Signaling), anti-phospho-c-Fos (1:1000) monoclonal antibody (5348, Cell Signaling), anti-phospho-CREB (1:1000) monoclonal antibody (9198, Cell Signaling) or anti-Actin (1:700) polyclonal antibody (sc-1615, Santa Cruz Biotechnology) at 4°C overnight.
  • In vitro migration was performed using a 48-Transwell microchemotaxis Boyden Chamber unit (Neuroprobe, Inc.).
  • MSCs 1.2 x 10 3 cells/well
  • TCM recombinant human AMF
  • rAMF recombinant human AMF
  • Both chambers were separated by 8 ⁇ m pore polycarbonate filters (Nucleopore membrane, Neuroprobe).
  • TCM were pre-incubated for 60 min with anti-AMF polyclonal antibody (sc-33777, Santa Cruz Biotechnology) or isotype control IgG.
  • BM-MSCs were incubated overnight (O.N.) with 1 ⁇ g/ml of rAMF in DMEM without FBS or DMEM without FBS as control.
  • the polycarbonate filters were previously incubated with 10 mg/ml type IV collagen (Sigma-Aldrich) for 18 h at 4°C; for MMP inhibition, BM-MSCs were preincubated with 1,10 phenantroline (0.5 or 1 mM) (Sigma-Aldrich). MSCs viability was not affected by 1,10 phenantroline (not shown). All the systems were incubated for 4 h at 37 °C in a 5% CO 2 humidified atmosphere. After that, the membrane was carefully removed and cells on the upper side of the membrane were scraped off with a blade.
  • Cells attached to the lower side of the membrane were fixed in 2% formaldehyde, and stained with 40,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich). Cells were counted using fluorescent-field microscopy and a 10X objective lens: the images captured in three representative visual fields were analyzed using CellProfiler software (www.cellprofiler.com), and the mean number of cells/field + SEM was calculated.
  • DAPI 40,6-diamidino-2-phenylindole dihydrochloride
  • AMF induced gelatinolytic activity in MSCs 5x10 4 cells were seeded in 24-well plates for 18 h. Cells were treated with 1 ⁇ g/ml of rAMF, TCM or serum-free DMEM as untreated control for 2 h; then, MSCs were washed with PBS and cultured in DMEM for 6 h before supernatants were collected. For blocking experiments, TCM were pre-incubated for 60 min with anti-AMF polyclonal antibody (sc-33777, Santa Cruz Biotechnology) or isotype control IgG. MMP2 activity was determined by zymography.
  • HMEC-1 For analyses of MSC adhesion to endothelial cells, 2x10 5 HMEC-1 were seeded in 96-well microplates and cultured for 1 day prior the assay. Coated wells were incubated for 5 minutes with 0.1 ml of 5x10 4 cells/ml of Fast-DiO labeled MSCs O.N. pretreated or not with 1 ⁇ g/ml rAMF. The cell suspension was discarded and the cells were fixed with 2% paraformaldehyde. Cells were counted using fluorescent-field microscopy and a 20X objective lens: the images captured in ten representative visual fields were analyzed using CellProfiler software (cellprofiler.com) and normalizing to untreated control.
  • CellProfiler software cellprofiler.com
  • RT-PCR Reverse Transcription-polymerase Chain Reaction
  • Total mRNA of BM-MSCs O.N. pretreated or not with 1 ⁇ g/ml rAMF was extracted using Trizol Reagent (Sigma-Aldrich Co., St. Louis, MO).
  • Trizol Reagent Sigma-Aldrich Co., St. Louis, MO
  • MSCs were 24 h starved before rAMF pre-treatment.
  • Total mRNA (4 ⁇ g) was reverse transcribed with 200 U of SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) using 500 ng of Oligo (dT) primers.
  • cDNAs were subjected to real-time polymerase chain reaction (qPCR) (Stratagene Mx3005p, Stratagene, La Jolla, CA, USA).
  • mRNA levels of metalloproteinase 3 MMP3, AMF receptor (AMFR), GDP dissociation inhibitor 2 (GDI-2), caveolin-1 (CAV-1) and caveolin-2 (CAV-2) were quantified by SYBR® Green (Invitrogen), using the following primer pairs:
  • PCR amplifications were carried out using a cycle of 95°C for 10 min and 45 cycles under the following parameters: 95°C for 30 sec, 58°C for 30 sec, 72°C for 1 min.
  • the temperature was increased from 60°C to 95°C at a rate of 2°C/min, and the fluorescence was measured every 15 sec to construct the melting curve. Values were normalized to levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; used as housekeeping) transcript (forward 5'-CATCTCTGCCCCCTCTGCTG -3' (SEQ ID NO: 12); reverse 5'-GCCTGCTTCACCACCTTCTTG-3' (SEQ ID NO: 13)). Data were processed by the ⁇ Ct method ( Livak KJ, Schmittgen TD. (2001) Methods 25(4):402-408 ).
  • the relative amount of the PCR product amplified from untreated cells was set as 1.
  • a non-template control (NTC) was run in every assay, and all determinations were performed in triplicate in three separated experiments.
  • HCC cells were seeded in 96-well culture tissue plates at 3x10 4 cells/cm 2 density for 1 day prior to the assay. Then cells were cultured with CM of BM-MSCs pre-treated with 1 ⁇ g/ml of rAMF for 48h. DMEM and CM of untreated BM-MSCs were used as control. Cell proliferation was evaluated by [3H]- thymidine incorporation assay. Each sample was assayed in sextuplicate and normalized to DMEM control.
  • Spheroid size was evaluated using inverted microscopy and a 4X objective lens: the images were captured and diameters determined using ImageJ software (National Institute of Health, NIH), finally spheroid volume was determined was calculated by the formula ⁇ /6 x larger diameter x (smaller diameter) 2 and expressed as arbitrary unity.
  • mice Six- to eight-week-old male nude BALB/c mice were purchased from CNEA (Comisión Nacional de Energ ⁇ a Atómica, Ezeiza, wholesome Aires, Argentina). The animals were maintained at our Animal Resources Facilities (School of Biomedical Sciences, Austral University) in accordance with the experimental ethical committee and the NIH guidelines on the ethical use of animals.
  • Subcutaneous model HuH7 cells (2 x 10 6 ) or HC-PT-5 cells (5 x 10 6 ) were inoculated subcutaneously (s.c.) into the right flank of nude mice.
  • rAMF recombinant human AMF
  • FI was performed using the Xenogen In Vivo Imaging System (IVIS; Caliper Life Sciences, Hopkinton, MA, USA). Mice injected with CMDiI-DiR-labeled MSCs were analyzed 1 h after MSC injection and every day until the experimental end point. Images represent the radiant efficiency and were analyzed with IVIS Living Image (Caliper Life Sciences) software. Regions of interest (ROI) were automatically drawn around the isolated organs to assess the fluorescence signal emitted.
  • IVIS In Vivo Imaging System
  • Results were expressed as average radiant efficiency in units of photons/second within the region of interest [p/s/cm 2 /sr] / [ ⁇ W/cm 2 ] or as total radiant efficiency in units of photons/second within the region of interest [p/s] / [ ⁇ W/cm 2 ].
  • MSCs Changes in the gene expression patterns in MSCs exposed to TCM derived from HCC samples were also analyzed. MSCs were exposed overnight to TCM or DMEM (as control) and studied using a microarray gene expression analysis with the aim to identify genes that were differentially expressed in MSCs exposed or not to TCM.
  • Table 3 shows 445 genes differentially expressed in MSC exposed to TCM from sample 1 in comparison with non-exposed cells.
  • Table 4 shows 511 genes differentially expressed in MSC exposed to TCM from sample 2 in comparison with non-exposed cells.
  • Table 5 shows 521 genes differentially expressed in MSC exposed to TCM from sample 4 in comparison with non-exposed cells.
  • Table 6 shows 511 genes differentially expressed in MSC exposed to TCM from sample 5 in comparison with non-exposed cells.
  • FIG. 1A shows the relative mRNA expression of up-regulated genes CTGF, CYR61, GJA1, SPARC, and AMFR.
  • Autocrine Motility Factor Receptor (AMFR) was up-regulated in MSCs exposed to all CM derived from HCC samples.
  • Figure 1B shows relative mRNA expression of down-regulated genes HSPA1A, HSP1B, and IGFBP3.
  • Recombinant AMF exerts a specific chemoatractant activity on MSCs from different sources
  • TCM tumor conditioned media
  • a 55 kDa soluble AMF was detected in CCM and TCM ( Figure 2A ).
  • MSCs from different sources were evaluated by in vitro migration assay with modified Boyden chambers as described in Example 1.
  • Human MSCs derived from bone marrow (BM-MSCs), perivascular umbilical cord region (Mesenchymal cells harvested from umbilical cord perivascular tissue), or adipose tissue (AT-MSCs) were used in a modified Boyden chamber assay.
  • the MSCs from the different sources migrated in a dose-dependent manner towards recombinant AMF ( Figure 2B-D ).
  • the most significant migration degree was shown in the dose ranging between 0.5 ⁇ g/mL and 1 ⁇ g/mL (p ⁇ 0.01) of rAMF for both BM-MSC and Mesenchymal cells harvested from umbilical cord perivascular tissue ( Figure 2B-C ), while AT-MSCs migrated better at 0.75 ⁇ g/mL of rAMF ( Figure 2D ).
  • rAMF concentration 5 ⁇ g/mL or 10 ⁇ g/mL were not capable of inducing migration neither in BM-MSCs nor in Mesenchymal cells harvested from umbilical cord perivascular tissue and AT-MSCs.
  • TCM were pretreated with polyclonal antibody against AMF (anti-AMF) to examine whether HCC tumor-secreted AMF was involved in MSC migration as described in the methods of Example 1.
  • anti-AMF polyclonal antibody against AMF
  • BM-MSCs showed a 40% reduction of migration in response to TCM derived from both HCC tumors.
  • a similar effect was observed in Mesenchymal cells harvested from umbilical cord perivascular tissue with a reduction of 30% and 40% in the response to TCM from HuH7 and HC-PT-5, respectively.
  • the reduction in AT-MSC migration potential was 30% and 20% towards TCM from HuH7 and HC-PT-5, respectively.
  • results show that AMF was secreted in the culture monolayers from HCC s.c tumors. Moreover, the results show for the first time that AMF produced by HCC is a chemoattractant factor for MSCs and induces migration of MSCs.
  • the migration was shown using MSCs from different sources (i.e., bone marrow (BM), perivascular cells from umbilical cord (Mesenchymal cells harvested from umbilical cord perivascular tissue) and adipose tissue (AT-MSCs)) and the MSCs from all of he tested sources exhibited migration towards AMF in a dose-dependent manner. 1 ⁇ g/ml of AMF was sufficient to induce MSCs migration.
  • BM bone marrow
  • perivascular cells from umbilical cord
  • AT-MSCs adipose tissue
  • MMPs matrix metalloproteinase
  • MMP3 mRNA level in MSCs was evaluated by qRT-PCR as described in Example 1.
  • MMP3 transcripts showed a 2.4-fold increase in BM-MSCs and Mesenchymal cells harvested from umbilical cord perivascular tissue, and 1.4-fold in AT-MSCs exposed to rAMF compared to unexposed cells ( Figure 3A ).
  • BM-MSCs stimulated with HCC CCM had increased MMP2 activity.
  • gelatinolytic activity corresponding to MMP2 was detected in supernatants from BM-MSCs and also from Mesenchymal cells harvested from umbilical cord perivascular tissue and AT-MSCs.
  • MMP2 activity was measured by zymography (as described in Example 1) in MSCs culture supernatant pre-stimulated with 1 ⁇ g/mL of rAMF or from un-stimulated cells as control to determine whether the induction of MMP2 was dependent on the presence of AMF in the TCM.
  • MMP2 activity was significantly enhanced when different sources MSCs were stimulated with rAMF ( Figure 3B ).
  • results show that MMP3 expression and MMP2 activity was induced in MSCs by rAMF.
  • rAMF increased the expression of mRNA MMP3 in MSCs.
  • results also show that AMF present in the TCM was, at least in part, responsible for the increased in MMP2 activity, which supports a critical role for AMF in MSC migration and invasion since blockage of AMF decreased MMP2 activity and inhibition of MMP2 decreased invasion in vitro.
  • AMF enhances BM-MSCs migration towards HCC by stimulating endothelial cell adhesion and modulating critical related genes
  • rAMF pretreatment induced a 40% increase in BM-MSCs migration to conditioned medium from ex vivo s.c. tumors (TCM) derived from HuH7 or HC-PT-5 cell lines. These results show that rAMF pretreatment influenced migration of MSCs towards TCM.
  • rAMF treatment induced the expression of AMFR, and the proteins involved in AMF-AMFR signaling pathways such as JNK, p-JNK, c-Fos, p-c-Fos and p-CREB ( Figure 4E ).
  • HUCPVCs presented higher migration and adhesion than BM-MSCs
  • IGFBP3 insulin-like growth factor-binding protein 3
  • MSCs will be engineered to express oncolytic virus expressing or not anti-tumor genes (including e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (e.g., interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), or a suicide gene (e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha).
  • anti-tumor genes including e.g., an interferon (e.g., interferon a, interferon ⁇ ), an interleukin (e.g., interleukin 1, interleukin 12), a chemokine (e.g., CX3CL1), or a suicide gene (e.g., thymidine kinase, IL-12, IFN-gamma, TNF-alpha).
  • infected MSCs will be stimulated in culture with rAMF for about 18 h and systemically injected (5x10 5 ) in HCC, colorectal cancer, and/or breast cancer tumor-bearing mice. Tumor growth will be assessed by calliper and tumor volume (mm 3 ) will be calculated using the formula ⁇ /6 x larger diameter x (smaller diameter) 2 .

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Claims (8)

  1. Procédé permettant (1) d'augmenter la migration d'une cellule souche mésenchymateuse (CSM) jusqu'à une tumeur ou une cellule tumorale ou (2) d'augmenter l'adhérence d'une CSM à une cellule endothéliale, le procédé comprenant la stimulation de la CSM avec un facteur autocrine de motilité recombinant (rAMF) in vitro, dans lequel la CSM comprend un agent thérapeutique.
  2. Procédé selon la revendication 1, dans lequel la cellule endothéliale est une cellule endothéliale vasculaire.
  3. Procédé selon la revendication 1 ou 2, dans lequel la tumeur ou la cellule tumorale exprime un AMF endogène.
  4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel la source de la CSM est sélectionnée dans le groupe constitué de la moelle osseuse, du tissu adipeux, et du cordon ombilical ; optionnellement dans lequel la CSM de cordon ombilical est récoltée à partir du tissu périvasculaire du cordon ombilical humain.
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel l'agent thérapeutique est un gène antitumoral recombinant ou un virus oncolytique manipulé pour exprimer un gène antitumoral recombinant.
  6. Procédé selon la revendication 5, dans lequel le gène antitumoral est sélectionné dans le groupe constitué d'un interféron, d'une interleukine, d'une chimiokine, d'un gène de suicide, et de n'importe quelle combinaison de ceux-ci.
  7. Procédé selon la revendication 5, dans lequel le gène antitumoral est sélectionné dans le groupe constitué de l'interféron α, de l'interféron β, de l'interleukine 1, de l'interleukine 12, de CX3CL1, de la thymidine kinase, de l'IL-12, de l'IFN-gamma, du TNF-alpha, et de n'importe quelle combinaison de ceux-ci.
  8. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel la CSM comprend en outre un récepteur de l'AMF recombinant.
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CA3059634A1 (fr) 2017-04-13 2018-10-18 Senti Biosciences, Inc. Immunotherapie anticancereuse combinatoire
EP3810159A4 (fr) * 2018-05-04 2022-01-19 Figene, LLC Libération de fibroblastes d'agents inhibiteurs de tumeur
EP3866813A4 (fr) 2018-10-17 2022-08-03 Senti Biosciences, Inc. Immunothérapie anticancéreuse combinatoire
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US20040258669A1 (en) * 2002-11-05 2004-12-23 Dzau Victor J. Mesenchymal stem cells and methods of use thereof
US20070128174A1 (en) * 2005-09-21 2007-06-07 Kleinsek Donald A Methods and compositions for organ and tissue functionality
WO2008075045A1 (fr) * 2006-12-18 2008-06-26 Cartela R & D Ab AGENTS DE FIXATION À LA SOUS-UNITÉ α-11 DE L'INTÉGRINE, ET SES UTILISATIONS
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US11173180B2 (en) 2021-11-16
AR097570A1 (es) 2016-03-23
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EP3047275A1 (fr) 2016-07-27
WO2015035235A1 (fr) 2015-03-12
US20190022143A1 (en) 2019-01-24

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